The CCAAT/enhancer binding protein β (C/EBPβ) cooperates with NFAT to control expression of the calcineurin regulatory protein RCAN1-4

Regulator of calcineurin 1 (RCAN1) inhibits the protein phosphatase calcineurin and is required for appropriate immune responses, synaptic plasticity, vascular tone, angiogenesis, and cardiac remodeling. Expression of the RCAN1-4 isoform is under the control of the calcineurin-responsive transcription factor NFAT. Typically, NFATs act in cooperation with other transcription factors to achieve maximal activation of gene expression. In this study, we identify the CCAAT/enhancer binding protein β (C/EBPβ) as an NFAT binding partner that cooperates with NFAT to regulate RCAN1-4 expression. Numerous C/EBPβ binding sites are conserved in theRCAN1-4 proximal promoter. Overexpression of C/EBPβ increased activity of both the endogenous mouse Rcan1-4 gene and a human RCAN1-4 luciferase reporter. Binding of C/EBPβ to multiple sites in the promoter was verified using electrophoretic mobility shift assays and chromatin immunoprecipitation.Adirect interaction between C/EBPβ and NFAT was demonstrated by coimmunoprecipitation of proteins and complex formation at NFAT-C/EBPβ composite sites. Depletion of endogenous C/EBPβ decreased maximal activation of RCAN1-4 expression by calcineurin, whereas inhibition of calcineurin did not alter the ability of C/EBPβ to activate RCAN1-4 expression. Together, these findings suggest that calcineurin/NFAT activation ofRCAN1-4 expression is in part dependent upon C/EBPβ, whereas activation by C/EBPβ is not dependent on calcineurin and may provide a calcineurin-independent pathway for regulating RCAN1-4 expression. Importantly, nuclear localization, C/EBPβ DNA binding activity and occupancy of the Rcan1-4 promoter increased in mouse models of heart failure demonstrating in vivo activation of this pathway to regulate Rcan1-4 expression and ultimately shape the dynamics of calcineurin-dependent signaling.